Polymerase chain reaction (PCR) exponentially amplifies DNA using temperature cycling to generate millions of copies of a target nucleic acid sequence from limited starting amounts of nucleic acids. Generally during PCR, target DNA is amplified by denaturing the DNA, annealing short primers to resulting single strands at specific sites (e.g., sequences flanking the target site) and extending the primers using a thermostable polymerase to generate new copies of double-stranded DNA complementary to the target. Typically, the PCR reaction mixture is repeatedly cycled (e.g., 20-50 times) from high temperatures (e.g., >90° C.) to denature the DNA to lower temperatures (e.g., between about 37° C. to 70° C.) for primer annealing and extension. Primer annealing and extension can be performed at the same or different temperatures.
In most automated PCR instruments, the reaction mixture is placed within a disposable plastic tube which is closed with a cap and placed within a metal heat-conducting sample block. The sample block is in communication with a processor which controls the cyclical heating of the block. As the metal block changes temperature, the reaction mixture is exposed to similar changes in temperature. Generally, PCR instruments provide a heating element at the bottom of the sample block in the form of a Peltier thermoelectric device or a thin foil heating element (MINCO brand, Minneapolis, Minn.) or alternatively supply a heated or cooled fluid through channels machined into the sample block. The use of these types of heating devices can result in delays in transferring heat from the sample block to the reaction mixture which may not be the same for all samples. Thus, both the efficiency and uniformity of amplification of nucleic acids within the samples can suffer as a consequence.
Evaporation from the PCR mixture during thermal cycling also can decrease the uniformity of amplification. Since the reaction mixture generally occupies only a fraction of a sample tube, this leaves a volume of air (known as “head space”) above the reaction mixture into which the reagents of the reaction can evaporate and subsequently condense. Various strategies have been used to minimize this problem. For example, a hydrophobic material such as mineral oil can be layered onto the PCR reaction mixture. The hydrophobic material floats on the reaction mixture and completely covers the surface of the reaction mixture, preventing evaporation from the mixture and condensation onto the side walls of the sample tube. A variant method relies on the use of a small solid wax ball or grease that melts at denaturing temperatures and which can be used to cover the surface of the reaction mixture (see, e.g., as described in U.S. Pat. No. 5,411,876). A commercially available wax ball used for this purpose is AMPLIWAX available from PERKIN-ELMER, Norwalk, Conn., U.S.A. However, adding hydrophobic materials or wax balls to the reaction mixture is both time consuming and increases the probability of sample contamination.
Another strategy to prevent or minimize sample evaporation and condensation includes the use of an external heater which is in proximity with the sample block. For example, the Stratagene Hot Top Assembly™ hot top for the RoboCycler® thermocycler provides a mechanism for heating the top of sample tubes placed in a sample block while the thermocycler's heating element heats the bottom of the sample block. The Hot Top Assembly™ significantly reduces condensation but a slight ring of condensation above the PCR reaction mixture in sample tubes may still be observed.